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Applied Biochemistry andBiotechnologyPart A: Enzyme Engineering
andBiotechnology ISSN 0273-2289Volume 172Number 6 Appl Biochem
Biotechnol (2014)172:2932-2944DOI 10.1007/s12010-014-0731-7
Thermokinetic Comparison of Trypan BlueDecolorization by Free
Laccase and FungalBiomass
N.N.A.Razak & M.S.M.Annuar
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1 23
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Thermokinetic Comparison of Trypan Blue Decolorizationby Free
Laccase and Fungal Biomass
N. N. A. Razak & M. S. M. Annuar
Received: 8 February 2013 /Accepted: 6 January 2014 /Published
online: 25 January 2014# Springer Science+Business Media New York
2014
Abstract Free laccase and fungal biomass from white-rot fungi
were compared in thethermokinetics study of the laccase-catalyzed
decolorization of an azo dye, i.e., Trypan Blue.The decolorization
in both systems followed a first-order kinetics. The apparent
first-order rateconstant, k1, value increases with temperature.
Apparent activation energy of decolorizationwas similar for both
systems at 22 kJ mol1, while energy for laccase inactivation was18
kJ mol1. Although both systems were endothermic, fungal biomass
showed higherenthalpy, entropy, and Gibbs free energy changes for
the decolorization compared to freelaccase. On the other hand, free
laccase showed reaction spontaneity over a wider range
oftemperature (T=40 K) as opposed to fungal biomass (T=15 K).
Comparison of entropychange (S) values indicated metabolism of the
dye by the biomass.
Keywords Azo dye . Decolorization . Laccase . Thermodynamic .
Kinetics . White-rot fungi
Introduction
Synthetic dyes are used in industries such as textile, paper,
pharmaceutical, cosmetics, andfood industries [1]. Due to
large-scale production and extensive applications, synthetic
dyescan cause considerable environmental pollution and serious
health-risk factors [2]. They areretained on the substrates by
physical adsorption, chemical interactions with metals and
salts,mechanical retaining, and solution or via covalent bonding.
Decolorization of these dyes byphysical or chemical adsorption and
precipitation methods are usually time-consuming andmostly
ineffective [3]. Furthermore, these methods cause accumulation of
the dye as sludgeand create a disposal problem later on. Hence,
extensive efforts are being focused on biologicalprocesses, as they
are relatively cost-effective and environmentally friendly provided
that thedyes are metabolizable.
Appl Biochem Biotechnol (2014) 172:29322944DOI
10.1007/s12010-014-0731-7
N. N. A. Razak :M. S. M. Annuar (*)Institute of Biological
Sciences, Faculty of Science, University Malaya, 50603 Kuala
Lumpur, Malaysiae-mail: [email protected]
M. S. M. AnnuarCentre for Research in Biotechnology for
Agriculture (CEBAR), University of Malaya, 50603 KualaLumpur,
Malaysia
Author's personal copy
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Laccase (EC 1.10.3.2; benzenediol/oxygen oxidoreductase) has
been the focus of manystudies due to its reaction versatility and
potential biotechnological applications. It is amultinuclear
copper-containing enzyme which uses molecular oxygen to oxidize a
widevariety of aromatic compounds [4]. They are widely distributed
in nature, originating fromplants, insects, bacteria, and
especially fungi. In fact, they are primarily found in fungi
andinvolved in lignin degradation, pigment biosynthesis, and
detoxification of lignin-derivedproducts. White-rot fungi produce
three main extracellular enzymes involved in ligninolysisviz.
laccase, lignin peroxidase, and manganese peroxidase [5].
Laccase has been indicated as capable of oxidizing amines,
phenolic and nonphenolic lignin-related compounds, and also highly
recalcitrant environmental pollutants such as synthetic dyesby a
complex ligninolytic enzymatic system [6, 7]. Due to their
versatility and adaptability,bioremediation using laccase has been
extensively studied for water remediation. Studies haveshown that
laccase from Tramates versicolor [8], Trametes hirsuta [9],
Trametes villosa [10],Trametes trogii [11], Trametes modesta [12],
Coriolus versicolor [13], Pcynoporus sanguineus[14], Pycnoporus
cinnabarinus [15], and Pleurotus ostreatus [16] could be used for
the degra-dation of a diverse chemical structure of dyemolecules.
Azo dyes are the major group of syntheticdyes, which are produced
in large quantities and have become a major source of concern
forchromophoric pollution. Besides causing esthetic damage, they
are also toxic and carcinogenic[17]. The decolorization of azo dyes
by white-rot fungi has been reported [1823]. However,comparative
studies on the thermodynamics and kinetics aspects of azo dyes
decolorization byfree laccase and fungal biomass from white-rot
fungi are lacking. Understanding these aspects isimportant for
rational design of treatment process for synthetic dyes run offs.
Therefore, in thiswork, the decolorization thermokinetics of Trypan
Blue, an azo dye by free laccase enzyme wasinvestigated, and this
was subsequently compared to the thermokinetics of decolorization
of thesame dye by fungal biomass of a white-rot fungus, i.e.,
Pycnoporus sanguineus.
Materials and Methods
Optimization Parameters for Dye Decolorization
The first part of this study investigated the effects of
selected variables on the decolorization ofTrypan Blue by free
laccase, i.e., enzyme concentration, reactant concentration, pH,
andtemperature.
Commercial laccase from Trametes versicolor (Sigma-Aldrich) with
specific activity0.8 U mg1 was used in this experiment without
further purification. Laccase was preparedby dissolving the enzyme
in 50 mM sodium citrate buffer (pH 4.8). The desired
laccaseconcentrations (0.008, 0.01, 0.03, 0.05, 0.07, 0.10 U ml1)
were obtained by stock dilution.Syringaldazine
(4-hydroxy-3,5-methoxybenzaldehyde; Sigma) was used as the
substrate forthe enzyme activity assay. Of the syringaldazine, 1 mM
stock was prepared by dissolving it in99.5 % ethanol and the
solution was kept at 4 C. Syringaldazine concentrations (0.02,
0.06,0.10, 0.20, 0.30, 0.40, and 0.50 mM) were prepared from the
stock solution by dilution with50 % ethanol. The syringaldazine
stock solution was warmed to room temperature before use.To
investigate the effect of pH, 50 mM sodium citrate buffer was
prepared for a pH range of 3to 6. Selection of different pH of
sodium citrate buffer was based on the nearest pKa values ofthe
citric acid. By referring to the pKa values of citric acid (3.13,
4.76, and 6.40), pH of sodiumcitrate buffer chosen were 3, 4, 4.8,
5, and 6. Similarly, a series of experiments were performedto
investigate the effect of temperature within the range of 4 to 80 C
on laccase activities.Three independent replicates were made for
every experiments conducted.
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Enzyme Assay
Laccase activity was measured by monitoring the rate of
oxidation of syringaldazine by theenzyme at 251 C. Of the laccase
solution, 0.2 mL was mixed with 3.0 ml of 50 mM citratebuffer (pH
4.8) in a cuvette. Of the syringaldazine, 0.2 mL of 0.1 mM was
added and gentlymixed, and the absorbance was measured immediately
at 525 nm for 10 min using UV/VISscanning spectrophotometer Jasco
V-630 (Japan). Total reaction volume was 3.4 mL. Laccaseactivity
was calculated as shown in Eq. (1),
Laccase activity UL
Abst
l total assay enzyme
enzyme sample1
whereAbs is the change of absorbance at 525 nm, t is the
incubation time (10 min), is theextinction coefficient for
syringaldazine (525=65,000 M
1 cm1), and l cm is the light pathlength (1 cm). One unit
activity is defined as the amount of laccase that oxidizes 1 mol
ofsyringaldazine per minute.
Trypan Blue Decolorization Assay
Trypan Blue (C34H24N6O14S4Na4, molar mass of 961 Da) was used
throughout the experi-ment. A stock solution of Trypan Blue was
prepared by dissolving 60 mg of the dye in 1 L of50 mM of sodium
citrate buffer (pH 4.8) and stored in an amber bottle to protect it
from directsunlight. The solution was kept at 4 C and was diluted
in appropriate concentrations (10, 20,30, 40, and 50 mg L1) before
use.
Batch decolorization process was initiated by adding laccase to
100 mL dye solution inErlenmeyer flasks, which contained different
concentrations of Trypan Blue. Initial laccaseactivity was
determined at room temperature (251 C) to ensure that all flasks
had similarlevel of enzyme activities. The final enzyme
concentration was 301 U L1. The reactionflasks were incubated on a
rotary shaker incubator at 150 rpm and were monitored at
intervalstime for 48 h at different temperatures (288, 298, 303,
308, 318, and 328 K). Controlexperiments with heat-denatured enzyme
(100 C, 30 min) were also conducted in parallel.The assays were
done in triplicates.
Dye concentration was routinely measured using spectrophotometer
at 597 nm. Thecalibration of Trypan Blue concentration fitted the
following equation:
A597 0:0244Cdye 2
where A597 was the absorbance of the solution at 597 nm and Cdye
was the concentration of thedye in milligram per liter. Equation
(2) had a regression coefficient of 0.9999, which indicatedthat the
assay model was reliable to determine the dye concentration with
acceptable precision.Equation (2) was applied over a concentration
range of 0 to 60 mg L1.
Calculations
Trypan Blue Decolorization
Residual dye concentration was measured at regular intervals for
up to 48 h. The fraction of thedye decolorized was calculated using
Eq. (3).
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Mdecolorized M initialM residualM initial 3
where Mdecolorized is the fractional percentage of Trypan Blue
decolorized, Minitial is the initialdye concentration, and
Mresidual is the remaining dye concentration in the solution.
Rate of Dye Decolorization
Volumetric rate of dye decolorization (rvol, in milligram per
liter per hour) was calculated usingEq. (4):
rvol Ct 4
where C (in milligrams per milliliter) is the change in dye
concentration over the timeinterval t (in hours).
Activation Energy
The activation energy (Ea, in joule per mole) of decolorization
process was calculated using thelinearized Arrhenius equation as
shown in Eq. (5):
lnk01 lnA
Ea
RT5
where A is the frequency factor, R is the gas constant (8.3145 J
mol1 K1), and T is theabsolute temperature (in Kelvin). To
calculate k1, the volumetric rate of dye decolorization(Eq. 4) was
plotted against different initial dye concentrations for each
temperature tested (288,298, 303, 308, 318, and 328 K). The rate
constant k1 was obtained from the slope of theresulting linear
plot.
Thermodynamic Parameters
The decolorization reaction is assumed to be at equilibrium
state when the remaining dye insolution showed no further changes
in concentration over time at a particular temperature.
Thetransition of the dye color was also considered a one-step
process, thus the apparent equilib-rium constant Kapp was
calculated as follows:
Kapp P eqS eq
6
where Kapp is the apparent equilibrium constant, [P]eq is the
concentration of dye that has beendecolorized at equilibrium
([P]eq=initial dye concentrationdye concentration remaining
atequilibrium), and [S]eq is the residual dye concentration at
equilibrium.
Kapp was measured at various temperatures. The temperature
dependence of Kapp isexpressed according to vant Hoff equation as
follows:
lnKapp HRT SR
7
where apparent enthalpy (H) is vant Hoff enthalpy (in joule per
mole) and entropy (S) isentropy (in joule per mole per Kelvin).
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At constant pressure and temperature, Gibbs free energy change
(G, in joule per mole) forthe reaction at nonstandard conditions
was calculated using the following equation:
G HTS 8Gibbs free energy at standard condition (G, in joule per
mole) was calculated as follows:
G GRT lnKapp 9where Kapp is the apparent equilibrium constant at
standard conditions.
Results and Discussion
Optimized Parameters for Dye Decolorization
Laccase Concentration
A linear relationship was observed between initial rate of
reaction and the enzyme concentra-tion within 8 to 70 U L1 ranges.
Within 10 s from the start of the assay, the percentage ofsubstrate
(dye) converted to product was calculated so that only 5 % or less
of dye decolorizedwithin this period. This was done to ensure that
at any enzyme concentration tested, thesubstrate was still in
excess amount and thus give accurate estimation for the initial
rate ofreaction. From the straight line obtained, 30 U L1 laccase
was chosen as the optimum laccaseconcentration for
decolorization.
Effect of Reactant Concentration
A rectangular hyperbolic graph was obtained when initial rate of
reaction was plotted againstsyringaldazine concentration (Fig. 1).
From the graph, it was clear that at lower
syringaldazineconcentrations ranging from 0.02 to 0.10 mM, the rate
of reaction is directly proportional tothe syringaldazine
concentration. However, at higher substrate concentrations
(0.200.50 mM)the initial rate of reaction was constant. This
indicated that the oxidation activity was at themaximum and the
active sites of the enzyme were virtually saturated with the
substrates. Anyfurther addition of the substrate will not alter the
rate of reaction. Thus, 0.20 mM of
Concentration of Syringaldazine (mM)
0.0 0.2 0.4 0.6
Initi
al r
ate
of r
eact
ion
(m
ol m
Lm
in-1
-1)
0.00
0.01
0.02
0.03
0.04
0.05Fig. 1 Initial rate of reaction fordifferent
syringaldazineconcentrations
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syringaldazine was taken to be the optimum reactant
concentration for laccase activity assay.
Effect of Buffer pH
As shown in Fig. 2, it was clear that pH significantly
influenced the laccase activity. Within apH range of 4.8 and 5, the
laccase activities were very similar. However, sharp decline
inactivities were observed at pH 3 and 6 (Fig. 2), which might be
resulted from improper ionicform of the laccases active site and
the substrate. pH stability of an enzyme depends on manyfactors
including ionic strength and chemical nature of the buffer. Based
on the ANOVA test,all pH showed significant differences except for
pH 4.8 and 5. Both pHs exhibited highestinitial rate of reaction,
so either pH (i.e., pH 4.8 or 5) is expected to be similarly
suitable for theenzyme. For decolorization experiment, 50 mM sodium
citrate buffer at pH 4.8 was employed.
Effect of Temperature on Laccase Activities
Comparable laccase activities were observed within temperature
range of 4 to 40 C, i.e.,124 U L1 (
-
Table 1 showed the maximum decolorization (63 %) was observed
after 48 h of incubation.Hence, the optimum temperature for Trypan
Blue decolorization by laccase was observed to bewithin the range
of 298 to 308 K since comparable results were obtained. According
toSadhasivam et al. [24], decolorization of Trypan blue by laccase
from Trichoderma harzianumreached about 33 % with laccase alone and
67 % in the presence of synthetic redox mediator
1-hydroxybenzotriazole at lower dye concentrations than were used
in this study. In otherstudies, DSauza et al. [25] and Baldrian et
al. [26] reported that Trypan Blue was decolorizedonly up to 25 %
by marine fungus NIOCC #2a and 42 % by a white-rot fungus
Daedaleaquercina. It is clear that the maximum percentage of Trypan
Blue decolorization in this studywas considerably higher than in
the previously reported literature. This was despite the
higherconcentration of Trypan Blue used in this study, and the
absence of any type of added syntheticredox mediator(s), which
could be used to enhance the rate of decolorization.
The lowest percentage of Trypan Blue decolorization was observed
at the highest temper-ature used, i.e., 328 K (Table 1). It was
observed that for any dye concentration investigated atthis
temperature, decolorization activities could be sustained only for
the first 6 h. After that,the activities decreased and no further
change in the concentration of the residual dye wasobserved. These
results were in accordance with Monteiro et al. [27] which found
laccase fromT. versicolor was stable for 6 h at 55 C (328 K).
Table 1 Percentage of Trypan Blue decolorization at different
temperatures
Concentration of TrypanBlue (mg L1)
Percentage of decolorization (%) at equilibrium at different
temperatures (K)
288 298 303 308 318 328
10 53 62 62 60 57 23
20 55 62 63 63 57 30
30 53 62 63 62 58 32
40 53 62 62 60 59 30
50 53 61 62 61 59 28
Standard deviation of the triplicate measurements was
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Apparent First-Order Rate Constant, k1
For the determination of apparent rate constant (k1), the graph
of the amount of dyedecolorized against 48 h of incubation time at
various concentration of dye was plotted foreach temperature tested
(data not shown). Then, from the slope of the graph, volumetric
rate ofdecolorization was calculated using Eq. (4). Then, the
volumetric rates of decolorization wereplotted against initial
concentrations of Trypan Blue in order to estimate the k1 of the
dyedecolorization (Table 2).
It was observed that the volumetric decolorization rate is
directly proportional to the initialdye concentration (1050 mg L1)
at any given temperature (288328 K; Fig. 4). For the freelaccase
system studied, the volumetric rate for every initial dye
concentration tested showed alinear increase with temperature.
Similar observation was made in the fungal biomass (pellet)system
investigated earlier [28]. For the free enzyme system, highest
volumetric rate wascalculated at 14.1 mg L1 h1 for the highest
initial dye concentration (50 mg L1) at 308 K.The apparent kinetics
of the decolorization followed a first-order behavior. As the
temperatureincreases, the slope of the line became steeper, which
reflected the increase in k1 values(Table 2). A higher k1 values
implies faster dye decolorization while a decrease in k1 value
isattributed to inactivation of laccase enzyme at higher
temperatures (318328 K).
Decolorization of dyes by Trametes spp. is shown to be following
a first-order kinetics inprevious studies. For example, T. modesta
showed an apparent first-order kinetics for thedecolorization of
various azo dye including CI Acid Orange 5, CI Acid Orange 52, CI
DirectBlue 71, CI Reactive Black 5, Orange 16, and CI Reactive
Orange 107 [18]. First-orderdecolorization kinetics are also
observed during the decolorization of an azo dye, Amaranth byT.
versicolor [29], in decolorization of both azo and anthraquinone
dye by laccase andmanganese peroxidase from T. versicolor [30] and
in the decolorization of Amaranth, ReactiveBlack 5, Reactive Blue
19, and Direct Black 22 by alginate-immobilized T. versicolor
[8].
Apparent Activation Energy of Decolorization
Based on k1 values obtained at different temperatures (Table 2),
the apparent activation energy(Ea) for the decolorization was
estimated using Arrhenius plot (Eq. 5). A plot of the ln k1
vs.reciprocal temperature was obtained with a regression
coefficient of 0.9874 (data not shown).From the slope of the line,
the Ea value was calculated at 21 kJ mol
1, which is consistent withactivation energy of enzyme-catalyzed
reactions, i.e., 1684 kJ mol1. This value is almost
Table 2 First-order rate constants for the decolorization at
different temperatures
Temperature (K) Volumetric rate (mg L1 h1) at different
concentrations ofTrypan Blue (mg L1)
Apparent first-order rateconstant, k1 (h
1)
10 20 30 40 50
288 1.4 3.2 4.7 6.4 8.1 0.160.009
298 1.7 4.0 5.9 8.2 10.2 0.200.007
303 2.3 4.9 7.2 10.0 12.3 0.250.003
308 2.8 5.6 8.3 11.1 14.1 0.280.005
318 2.0 4.2 6.2 8.6 10.8 0.210.003
328 1.9 3.7 6.0 7.4 7.7 0.170.007
Standard deviation of the triplicate measurements was
-
identical to the decolorization of Trypan Blue using
self-immobilized fungal biomass ofP. sanguineus previously reported
with Ea value 23 kJ mol
1 [28]. Other reported activationenergy for laccase was 21.175
kJ mol1 for phenol polymerization [31], 57 kJ mol1 for 1-napthol
polymerization [32], and 44.8 kJ mol1 for chlorophenol degradation
[33], respective-ly. In this study, laccase-mediated decolorization
activities were significantly reduced at 318 to328 K. Thus, using
Eq. (5), apparent inactivation energy for laccase catalyzed
decolorizationof Trypan Blue, Ed was calculated at 18 kJ mol
1.
Thermodynamics of Decolorization
Employing Vant Hoff equation (Eq. 7),H andS were determined from
the slope and the yintercept of the straight line (Fig. 5). A plot
of ln Kapp against reciprocal temperature fitted astraight line
with a regression coefficient of 0.9446 (Fig. 5).
Kapp refers to the apparent equilibrium constant (Eq. 6) for
decolorization process, whichwas calculated for the highest dye
concentration, i.e., 50 mg L1 at each temperature tested
Initial Concentration of Trypan Blue (mg L-1)
0 10 20 30 40 50 60
Rat
e of
dec
olor
izat
ion
(mg
L-1
h-1 )
0
2
4
6
8
10
12
14
16
298 K298 K303 K308 K318 K328 K
Fig. 4 Apparent first-order rate constant, k1 (in hours) as a
function of its initial concentration and temperature
1/T (K)0.00320 0.00325 0.00330 0.00335 0.00340 0.00345
0.00350
ln K
app
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.9446R
7044.45.13202
xy ==
+
Fig. 5 Vant Hoff plot fordecolorization of Trypan Blueby free
laccase
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(Table 3). The Kapp value increases with the increase in
temperature until 318 K and abovewhere it decreased.
From this work, H and S were calculated at 11 kJ mol1 and 39 J
mol1 K1, respec-tively. The positive value of H for the processes
implies the endothermic nature of thedecolorization system, whereas
the positive value ofS reflects a decolorization system that
isbecoming increasingly disordered as the temperature
increases.
The values of enthalpy and entropy changes obtained subsequently
were used in thecalculation of Gibbs free energy change, G using
Eq. (8). The G value indicates thedegree of spontaneity of the
decolorization process. A strong negative value reflects a
moreenergetically favorable process [34]. As shown in Table 3,
decolorization reaction occursspontaneously for all temperatures
studied (288328 K). Thus, the Trypan Blue decolorizationcatalyzed
by laccase is an energetically favorable process and spontaneous in
nature. More-over, it may also be noted that as more heat is
supplied to the system, the greater the tendencyof the system to
move towards decolorization. This is shown by strong increase in
the valueG as temperature increases. This could be explained as
follow; as temperature increases,more heat is absorbed (H) during
the process resulting in the increase of thermal energy ofthe
system which causes more energetically favorable interaction
between laccase and dyeresulting in increased chances of successful
decolorization.
From Table 3, the value of (GG) indicates the differences
between the energy changeoccurring during the reaction under the
conditions used and the energy change that wouldoccur if the
reaction took place under standard conditions (1 atm, 1 M, 298 K).
A graph ofGG as a function of absolute temperature (in Kelvin) and
initial dye concentration (inmilligram per liter) is shown in Fig.
6. An increase in energy surplus is clearly seen attemperature
range of 288308 K and a slight decrease at 318 K but is still
positive in thevalue of (GG). In contrast, huge energy deficit was
observed at the highest temperature,i.e., 328 K as indicated by the
steep plunge (Fig. 6).
A plot of theG for the decolorization reaction at various
temperatures is shown in Fig. 7.The G values were calculated using
Eq. (8) with the assumption that the H and Scalculated earlier for
free laccase system are constant within the temperature range
examined.It showed G=0 at approximately 280 K (or 7.28 C), which
means that at this temperature,the energy of the products and
reactants are at the equilibrium. As described before, values ofG
were negative for all temperatures studied (288328 K) showing that
Trypan Bluedecolorization by laccase is favored when temperature is
greater than 7.28 C. Meanwhile,extrapolated graph showed that the
reaction is nonspontaneous (i.e., G is positive) below7.28 C (280
K).
Energetics of Trypan Blue decolorization using fungal biomass
(pellets) of Pycnoporoussanguineus [28] was compared with the
results obtained in this study. P. sanguineus is known toproduce
laccase as its sole lignin degradation enzyme (phenoloxidase).
According to Fig. 7, a
Table 3 Gibbs free energy (G) of decolorization at 50 mg L1
Temp (C) Absolute temperature, T (K) Apparent Kapp G (J mol1) GG
(J mol1)
15 288 1.15 299 +33925 298 1.29 685 +64030 303 1.37 879 +79535
308 1.57 1,072 +1,16245 318 1.46 1,459 +1,00955 328 0.40 1,846
2,471
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linear function of G with temperature was observed for both free
laccase and fungal pelletsystems. For fungal pellet system, the H
and S values used in the calculation of G weretaken from the study
of [28]. It also illustrated that free laccase system has wider
temperaturerange of spontaneity (T=40 K) relative to fungal biomass
system (T=15 K). Moreover, freelaccase system was shown to be less
sensitive to temperature variation as compared to fungalbiomass
system. This implies that free laccase system can withstand
relatively huge perturba-tion in temperature with minimal effects
on the reaction spontaneity. In contrast, for fungalbiomass system
huge perturbation in temperature may drastically affect the
spontaneity of the
-4000
-3000
-2000
-1000
0
1000
2000
290295
300305
310315
320325
10
20
30
40
G -
G
(J)
Tempera
ture (K)
Concentration (m
g L -1)
-2500 -2000 -1500 -1000 -500 0
Fig. 6 Differences between Gibbs free energy change (G) and
Gibbs free energy change at standard condition(G) as a function of
initial dye concentration (in milligrams per liter) and temperature
(in Kelvin)
Temperature (K)
240 260 280 300 320 340
Gib
bs F
ree
Ene
rgy
(J)
-3000
-2000
-1000
0
1000
2000
3000
Free laccaseFungal pellet
(spontaneous)
G < 0
spontaneous)-(non
G > 01R
1097539.11x -y
0G
=
==
+
==+
Fig. 7 Comparison of Gibb free energy change as a function of
temperature between free laccase and fungalpellet systems
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reaction. H value for fungal biomass system (H=46 kJ mol1) was
shown to be fourfoldhigher as compared to free laccase system (H=11
kJ mol1). HugeH reflects more energyneed to be transferred to the
system to make it favorable. Furthermore, due to highS value
forfungal biomass system (S=146 J mol1 K1), it is hypothesized that
Trypan Blue wasmetabolized to byproduct(s) such as carbon dioxide,
thus explaining the high S value in thefungal biomass system
compared to the free enzyme system (S=39 J mol1 K1).
The study presented a strong support and rationalization for the
technical utilization ofbiological catalysts such as laccase and/or
its whole biomass system in the decolorization ofchromophore(s)
contaminated water bodies. While the free enzyme system is able to
toleraterelatively high temperature perturbation range as compared
to the whole biomass system in thedecolorization process, the
latter could be effectively use to metabolize the dye at the same
time.
Acknowledgments The authors acknowledge University of Malaya for
providing the research grants PG033-2013A, RP024-2012A, and
UM.C/625/1/HIR/MOHE/05.
Conflict of Interests All the authors of the submission declare
and clarify that we do not have a direct financialrelation with the
commercial identities mentioned in the paper that might lead to a
conflict of interest for any ofthe authors.
References
1. Ciullini, I., Tilli, S., Scozzafava, A., & Briganti, F.
(2008). Fungal laccase, cellobiose dehydrogenase, andchemical
mediators: combined actions for the decolorization of different
classes of textile dyes. BioresourceTechnology, 99(15), 70037010.
doi:10.1016/j.biortech.2008.01.019.
2. Forgacs, E., Cserhti, T., & Oros, G. (2004). Removal of
synthetic dyes from wastewaters: a review.Environment
International, 30(7), 953971. doi:10.1016/j.envint.2004.02.001.
3. Erkurt, E. A., nyayar, A., & Kumbur, H. (2007).
Decolorization of synthetic dyes by white rot fungi,involving
laccase enzyme in the process. Process Biochemistry, 42(10),
14291435. doi:10.1016/j.procbio.2007.07.011.
4. Litthauer, D., van Vuuren, M. J., van Tonder, A., &
Wolfaardt, F. W. (2007). Purification and kinetics of athermostable
laccase from Pycnoporus sanguineus (SCC 108). Enzyme and Microbial
Technology, 40(4),563568. doi:10.1016/j.enzmictec.2006.05.011.
5. Arora, D. S., & Gill, P. K. (2001). Effects of various
media and supplements on laccase production by somewhite rot fungi.
Bioresource Technology, 77(1), 8991.
doi:10.1016/s0960-8524(00)00114-0.
6. Balan, D. S. L., & Monteiro, R. T. R. (2001).
Decolorization of textile indigo dye by ligninolytic fungi.Journal
of Biotechnology, 89(23), 141145.
doi:10.1016/s0168-1656(01)00304-2.
7. Katuri, K. P., Venkata Mohan, S., Sridhar, S., Pati, B. R.,
& Sarma, P. N. (2009). Laccase-membrane reactorsfor
decolorization of an acid azo dye in aqueous phase: process
optimization.Water Resource, 43(15), 36473658.
doi:10.1016/j.watres.2009.05.028.
8. Ramsay, J., Mok, W. H. W., Luu, Y. S., & Savage, M.
(2005). Decoloration of textile dyes by alginate-immobilized
Trametes versicolor. Chemosphere, 61(7), 956964.
doi:10.1016/j.chemosphere.2005.03.070.
9. Domnguez, A., Couto, S. R., & Sanromn, M. . (2005). Dye
decolorization by Trametes hirsutaimmobilized into alginate beads.
World Journal of Microbiology and Biotechnology, 21(4), 405409.
doi:10.1007/s11274-004-1763-x.
10. Basto, C., Silva, C. J., Gbitz, G., & Cavaco-Paulo, A.
(2007). Stability and decolourization ability ofTrametes villosa
laccase in liquid ultrasonic fields. Ultrasonics Sonochemistry,
14(3), 355362. doi:10.1016/j.ultsonch.2006.07.005.
11. Zeng, X., Cai, Y., Liao, X., Zeng, X., Li, W., & Zhang,
D. (2011). Decolorization of synthetic dyes by crudelaccase from a
newly isolated Trametes trogii strain cultivated on solid
agro-industrial residue. Journal ofHazardous Materials, 187(13),
517525. doi:10.1016/j.jhazmat.2011.01.068.
12. Nyanhongo, G. S., Gomes, J., Gbitz, G. M., Zvauya, R., Read,
J., & Steiner, W. (2002). Decolorization oftextile dyes by
laccases from a newly isolated strain of Trametes modesta. Water
Research, 36(6), 14491456. doi:10.1016/s0043-1354(01)00365-7.
Appl Biochem Biotechnol (2014) 172:29322944 2943
Author's personal copy
http://dx.doi.org/10.1016/j.biortech.2008.01.019http://dx.doi.org/10.1016/j.envint.2004.02.001http://dx.doi.org/10.1016/j.procbio.2007.07.011http://dx.doi.org/10.1016/j.procbio.2007.07.011http://dx.doi.org/10.1016/j.enzmictec.2006.05.011http://dx.doi.org/10.1016/s0960-8524(00)00114-0http://dx.doi.org/10.1016/s0168-1656(01)00304-2http://dx.doi.org/10.1016/j.watres.2009.05.028http://dx.doi.org/10.1016/j.chemosphere.2005.03.070http://dx.doi.org/10.1007/s11274-004-1763-xhttp://dx.doi.org/10.1016/j.ultsonch.2006.07.005http://dx.doi.org/10.1016/j.ultsonch.2006.07.005http://dx.doi.org/10.1016/j.jhazmat.2011.01.068http://dx.doi.org/10.1016/s0043-1354(01)00365-7
-
13. Asgher, M., Batool, S., Bhatti, H. N., Noreen, R., Rahman,
S. U., & Javaid Asad, M. (2008). Laccasemediated decolorization
of vat dyes by Coriolus versicolor IBL-04. International
Biodeterioration &Biodegradation, 62(4), 465470.
doi:10.1016/j.ibiod.2008.05.003.
14. Pointing, S. B., & Vrijmoed, L. L. P. (2000).
Decolorization of azo and triphenylmethane dyes byPycnoporus
sanguineus producing laccase as the sole phenoloxidase. World
Journal of Microbiology andBiotechnology, 16(3), 317318.
doi:10.1023/a:1008959600680.
15. Camarero, S., Ibarra, D., Martnez, M. J., & Martnez, .
T. (2005). Lignin-derived compounds as efficientlaccase mediators
for decolorization of different types of recalcitrant dyes. Applied
and EnvironmentalMicrobiology, 71(4), 17751784.
16. Novotn, ., Rawal, B., Bhatt, M., Patel, M., aek, V., &
Molitoris, H. P. (2001). Capacity of Irpex lacteusand Pleurotus
ostreatus for decolorization of chemically different dyes. Journal
of Biotechnology, 89(23),113122.
doi:10.1016/s0168-1656(01)00321-2.
17. Srinivasan, C., Dsouza, T. M., Boominathan, K., & Redd,
C. A. (1995). Demonstration of laccase in thewhite rot
basidiomycete Phanerochaete chrysosporium BKM-F1767. Applied and
EnvironmentalMicrobiology, 61(12), 42744277.
18. Tauber, M. M., Gbitz, G. M., & Rehorek, A. (2008).
Degradation of azo dyes by oxidative processeslaccase and
ultrasound treatment. Bioresource Technology, 99(10), 42134220.
doi:10.1016/j.biortech.2007.08.085.
19. Chagas, E. P., & Durrant, L. R. (2001). Decolorization
of azo dyes by Phanerochaete chrysosporium andPleurotus sajorcaju.
Enzyme and Microbial Technology, 29(89), 473477.
doi:10.1016/s0141-0229(01)00405-7.
20. Wong, Y., & Yu, J. (1999). Laccase-catalyzed
decolorization of synthetic dyes. Water Resource, 33(16),35123520.
doi:10.1016/s0043-1354(99)00066-4.
21. Neifar, M., Jaouani, A., Kamoun, A., Ellouze-Ghorbel, R.,
& Ellouze-Chaabouni, S. (2011). Decolorizationof solophenyl red
3BL polyazo dye by laccase-mediator system: optimization through
response surfacemethodology. Enzyme Research.
doi:10.4061/2011/179050.
22. Yesilada, O., Asma, D., & Cing, S. (2003).
Decolorization of textile dyes by fungal pellets.
ProcessBiochemistry, 38(6), 933938.
doi:10.1016/s0032-9592(02)00197-8.
23. Wang, T.-N., Lu, L., Guo-Fu, L., Jun, L., Xu, T.-F., &
Zhao, M. (2011). Decolorization of the azo dyereactive black 5
using laccase mediator system. African Journal of Biotechnology,
10(75), 1718617191.doi:10.5897/AJB11.1780.
24. Sadhasivam, S., Savitha, S., & Swaminathan, K. (2009).
Redox-mediated decolorization of recalcitranttextile dyes by
Trichoderma harzianum WL1 laccase. World Journal of Microbiology
and Biotechnology,25(10), 17331741.
doi:10.1007/s11274-009-0069-4.
25. DSouza, D. T., Tiwari, R., Sah, A. K., & Raghukumar, C.
(2006). Enhanced production of laccase by amarine fungus during
treatment of colored effluents and synthetic dyes. Enzyme and
Microbial Technology,38(34), 504511.
doi:10.1016/j.enzmictec.2005.07.005.
26. Baldrian, P. (2004). Purification and characterization of
laccase from the white-rot fungusDaedalea quercinaand
decolorization of synthetic dyes by the enzyme. Applied
Microbiology and Biotechnology, 63(5), 560563.
doi:10.1007/s00253-003-1434-0.
27. Monteiro, M. C., & De Carvalho, M. E. A. (1998). Pulp
bleaching using laccase from Trametes versicolorunder high
temperature and alkaline conditions. Applied Biochemistry and
Biotechnology, 7072(1), 983993. doi:10.1007/BF02920208.
28. Annuar, M. S. M., Adnan, S., Vikineswary, S., & Chisti,
Y. (2009). Kinetics and energetics of azo dyedecolorization by
Pycnoporus sanguineus. Water, Air, and Soil Pollution, 202(1),
179188. doi:10.1007/s11270-008-9968-5.
29. Ramsay, J., Shin, M., Wong, S., & Goode, C. (2006).
Amaranth decoloration by Trametes versicolor in arotating
biological contacting reactor. Journal of Industrial Microbiology
and Biotechnology, 33(9), 791795.
doi:10.1007/s10295-006-0117-0.
30. Champagne, P.-P., & Ramsay, J. (2005). Contribution of
manganese peroxidase and laccase to dyedecoloration by Trametes
versicolor. Applied Microbiology and Biotechnology, 69(3), 276285.
doi:10.1007/s00253-005-1964-8.
31. engr, M., & Akta, N. (2009). A kinetic model development
for phenol removal via enzymatic polymer-ization. Hacettepe Journal
of Biology and Chemistry, 37(4), 295301.
32. Akta, N., iek, H., Tapnar nal, A., Kibarer, G., Kolankaya,
N., & Tanyola, A. (2001). Reactionkinetics for
laccase-catalyzed polymerization of 1-naphthol. Bioresource
Technology, 80(1), 2936. doi:10.1016/s0960-8524(01)00063-3.
33. Zhang, J., Liu, X., Xu, Z., Chen, H., & Yang, Y. (2008).
Degradation of chlorophenols catalyzed by laccase.International
Biodeterioration & Biodegradation, 61(4), 351356.
doi:10.1016/j.ibiod.2007.06.015.
34. Segel, I. H. (1976). Biochemical calculation: how to solve
mathematical problems in general biochemistry.USA: Wiley.
2944 Appl Biochem Biotechnol (2014) 172:29322944
Author's personal copy
http://dx.doi.org/10.1016/j.ibiod.2008.05.003http://dx.doi.org/10.1023/a:1008959600680http://dx.doi.org/10.1016/s0168-1656(01)00321-2http://dx.doi.org/10.1016/j.biortech.2007.08.085http://dx.doi.org/10.1016/j.biortech.2007.08.085http://dx.doi.org/10.1016/s0141-0229(01)00405-7http://dx.doi.org/10.1016/s0043-1354(99)00066-4http://dx.doi.org/10.4061/2011/179050http://dx.doi.org/10.1016/s0032-9592(02)00197-8http://dx.doi.org/10.5897/AJB11.1780http://dx.doi.org/10.1007/s11274-009-0069-4http://dx.doi.org/10.1016/j.enzmictec.2005.07.005http://dx.doi.org/10.1007/s00253-003-1434-0http://dx.doi.org/10.1007/BF02920208http://dx.doi.org/10.1007/s11270-008-9968-5http://dx.doi.org/10.1007/s11270-008-9968-5http://dx.doi.org/10.1007/s10295-006-0117-0http://dx.doi.org/10.1007/s00253-005-1964-8http://dx.doi.org/10.1007/s00253-005-1964-8http://dx.doi.org/10.1016/s0960-8524(01)00063-3http://dx.doi.org/10.1016/s0960-8524(01)00063-3http://dx.doi.org/10.1016/j.ibiod.2007.06.015
Thermokinetic Comparison of Trypan Blue Decolorization by Free
Laccase and Fungal BiomassAbstractIntroductionMaterials and
MethodsOptimization Parameters for Dye DecolorizationEnzyme
AssayTrypan Blue Decolorization Assay
CalculationsTrypan Blue DecolorizationRate of Dye
DecolorizationActivation EnergyThermodynamic Parameters
Results and DiscussionOptimized Parameters for Dye
DecolorizationLaccase ConcentrationEffect of Reactant
ConcentrationEffect of Buffer pHEffect of Temperature on Laccase
Activities
Effect of Temperature on Dye DecolorizationApparent First-Order
Rate Constant, k1Apparent Activation Energy of
DecolorizationThermodynamics of Decolorization
References